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Prevalence of celiac disease among first-degree relatives of Indian celiac disease patients
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Alimentary Tract
Prevalence of celiac disease among first-degree relatives of Indian celiac disease patients Asha Mishra a , Shyam Prakash a , Gurvinder Kaur b , Vishnubhatla Sreenivas c , Vineet Ahuja a , Siddhartha Datta Gupta d , Govind K. Makharia a,∗ a
Department of Gastroenterology and Human Nutrition, All India Institute of Medical Sciences, New Delhi, India Department of Transplant Immunology, All India Institute of Medical Sciences, New Delhi, India c Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India d Department of Pathology, All India Institute of Medical Sciences, New Delhi, India b
a r t i c l e
i n f o
Article history: Received 1 July 2015 Accepted 11 November 2015 Available online xxx Keywords: Family Haplotype HLA Seroprevalence
a b s t r a c t Background: Celiac disease, once thought to be uncommon in Asia, is now recognized in Asian nations as well. We investigated the prevalence of celiac disease in first-degree relatives of celiac disease patients followed in our centre. Methods: First-degree relatives were screened prospectively for celiac disease using questionnaire-based interview and anti-tissue transglutaminase antibody. Serology positive first-degree relatives underwent duodenal biopsies. Diagnosis of celiac disease was made based on positive serology and villous abnormality Marsh grade 2 or higher. Human leucocyte antigen DQ2/-DQ8 was also assessed in 127 first-degree relatives. Results: 434 first-degree relatives of 176 celiac disease patients were prospectively recruited; 282 were symptomatic (64.9%), 58 were positive for serology (13.3%). Seroprevalence was higher in female than in males (19% vs 8.5%; p = 0.001) and highest in siblings (16.9%) than parents (13.6%) and children (5.9%) of celiac patients (p = 0.055); 87.4% first-degree relatives were human leucocyte antigen-DQ2/-DQ8 positive. Overall prevalence of celiac disease was 10.9% amongst first-degree relatives. Conclusions: The prevalence of celiac disease in first-degree relatives of celiac disease patients was 10.9% in our cohort, and 87% had human leucocyte antigen-DQ2 or -DQ8 haplotype. All first-degree relatives of celiac disease patients should be screen for celiac disease even if asymptomatic or with atypical manifestations. © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Celiac disease (CD) is an immune-mediated enteropathy triggered by ingestion of gluten present in wheat and related prolamines present in rye and barley in genetically susceptible individuals carrying HLA-DQ2 and/or HLA-DQ8 haplotype [1]. Once thought to be uncommon, CD affects approximately 1% of the world’s population [2–4]. Even in Asian countries, where CD was thought to be a rare disease, recent studies – including one from our centre – suggest a prevalence of 1 in 330 to 1 in 96 in the general population in Northern India [5,6]. CD is now being observed
∗ Corresponding author at: Department of Gastroenterology and Human Nutrition, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. Tel.: +91 11 26588091/26546546; fax: +91 11 26588091/26588663. E-mail addresses: [email protected], [email protected] (G.K. Makharia).
in other Asian countries including China, Malaysia and Pakistan [7–10]. Since CD is a genetic disease, first-degree relatives (FDRs) of patients with CD are at higher risk of developing CD due to close genetic repertoire, which leads to higher genetic susceptibility [4]. Advent of celiac-specific antibodies (a reflection of adaptive immune response to gluten peptide) has revolutionized the case detection rate and has led to recognition of CD as a public health problem world over [11]. With the help of celiac-specific serologic tests, it is now possible to screen and detect CD not only the clinically apparent patients but also those who still have not developed any symptoms. With increasing use of screening and diagnostic tests along with increase in the awareness, CD has become one of the most common genetic disease. Because of the genetic susceptibility, both FDRs and seconddegree relatives are at a higher risk of developing CD than the general population. The prevalence of CD in FDRs vary widely from 5% to 38% [12–15]. The three studies from Asia, all the them from
http://dx.doi.org/10.1016/j.dld.2015.11.007 1590-8658/© 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mishra A, et al. Prevalence of celiac disease among first-degree relatives of Indian celiac disease patients. Dig Liver Dis (2015), http://dx.doi.org/10.1016/j.dld.2015.11.007
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the Northern part of India have also reported a prevalence of CD in FDRs to vary from 8.2% to 22% [16–18]. The risk of having CD in FDRs varies with the individual relationship with the index patient with CD. Several studies have suggested that siblings are at a higher risk of developing CD in comparison to parents and children [19,20,13]. The spectrum of symptoms in FDRs varies and a large number of them remain either asymptomatic and or mildly symptomatic. Only a proportion of them have full-blown symptoms. Since CD is now emerging in Asian countries, and there are two other studies from India with much smaller sample size in both the studies and inclusion of only siblings in one of the study [16,17]. Therefore, we conducted the present study to find out the prevalence of CD in the FDRs of patients with CD. 2. Methods 2.1. Subjects This prospective study was conducted at All India Institute of Medical Sciences, New Delhi, between May 2009 and September 2014. From the initial cohort of 540 patients with CD followed at our Celiac Disease Clinic, all FDRs were invited to be screened for CD. Four hundred and thirty four FDRs (54% of total live FDRs) of 176 index patients agreed to participate and were enrolled. This study was approved by the Ethics Committee of our Institution and written informed consent was obtained from each participant and parents or guardians for subjects aged less than 18 years. All FDRs were administered a questionnaire and they underwent a complete history and clinical evaluation. Five-millilitres of blood was collected and separated in a plain tube (2 ml) and in EDTA (3 ml). Plain tube sample was kept at room temperature for approximately an hour and then centrifuged at 2500 × g for 10 min. Supernatant was separated and was kept in a microcentrifuge tube at 20 ◦ C until analysis. DNA was isolated from the blood collected in the EDTA tubes. 2.2. IgA anti-tissue transglutaminase antibody (IgA anti-tTG Ab) FDRs were screened for CD using commercially available IgA anti-tTG Ab ELISA kits procured from AESKU Diagnostik, Wendelsheim, Germany (cut-off: 18 IU/ml). FDRs with a positive ant-tTG Ab were invited to undergo upper gastrointestinal endoscopy and duodenal biopsies.
The diagnosis of CD was made on the basis of the modified European Society of Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) criteria i.e., clinical features, presence of villous atrophy (villous abnormalities of modified Marsh grade ≥2) and unequivocal response to gluten-free diet [21]. 2.4. HLA genotyping The genotyping for HLA-DQ2 and -DQ8 was performed using reverse sequence specific oligonucleotides method (One Lambda, Inc., Thermo Fisher Scientific, USA). Readings were taken through Luminex X-Map technology (Luminex, Life Technologies, Inc). 2.5. Statistical analysis Quantitative variables were summarized as mean ± standard deviation. Qualitative were summarized as frequency (%). Qualitative variables were compared between those with a positive and negative anti-tTG Ab using Chi-square test. Quantitative variables were compared using student’s t test or Wilcoxon’s Ranksum test as appropriate. 3. Results Overall, 434 FDRs of 176 CD patients (54% of all living FDRs) were included in this study; 234 (53.9%) were males, mean age was 29.8 ± 15.9 years; 326 were over 18 years of age (75.1%). Relationships with patients were 159 siblings (36.6%), 191 parents (44%), and 84 children (19.3%). Four FDRs had previously been diagnosed with CD and were already following gluten-free diet. FDRs who agreed to participate were younger than those who did not consent (29.71 ± 15.95 vs 37.38 ± 16.7 years). However, there was no difference in gender distribution. 3.1. Symptoms Two hundred and eighty two FDRs (64.9%) had one or more symptoms; 140 had predominantly gastrointestinal symptoms (32.2%) and 253 had a combination of both gastrointestinal and extra intestinal symptoms (58.2%; Table 1); 152 were completely asymptomatic (35%). Hypothyroidism was present in 4. 3.2. Seroprevalence of CD
2.3. Endoscopy and mucosal biopsy examination Upper gastrointestinal endoscopy was performed and at least four to six biopsies were taken from the second or third part of the duodenum. A biopsy fragment was considered oriented when at least 3 crypts were oriented perpendicularly on the underlying muscularis mucosae. Biopsies were analyzed for mucosal changes by a histo-pathologist with special expertise in gastrointestinal pathology blinded to the clinical or serological results. The Modified Marsh grading system was used for grading mucosal changes: Grade 0, normal histology [a crypt to villous ratio of 1:3 was taken as normal]; Grade 1:increase of intraepithelial lymphocytes (IEL) >40/100 enterocytes (IELs were identified as dark round cells with high nucleus to cytoplasmic ratio, in comparison to the perpendicularly oriented cigar shaped vesicular nuclei of the mucosal epithelial cells); Grade 2: increased IELs along with crypt hyperplasia; Grade 3: increased IELs along with crypt hyperplasia and variable degrees of villous atrophy. A further semi-quantitative sub typing of the villous atrophy was performed as follows: grade 3a/mild villous atrophy [duodenal biopsies with a crypt to villous ration of >1:3 but <1]; grade 3b/moderate villous atrophy, C:V ratio of 1:1; grade 3c/severe villous atrophy, C:V ratio of >1.
Overall, 58 FDRs belonging to 44 index patient families tested positive for anti-tTG Ab (13.3%). This meant approximately 25% (one in 4) of families had at least one anti-tTG Ab positive FDR. Five families of index CD patients had 2 or more anti-tTG Ab positive FDRs. CD seroprevalence was higher in female than in male FDRs (19% vs 8.5%; p = 0.001). The seroprevalence was 16.9% in siblings vs 13.6% in parents and 5.9% in children (p = 0.055) of CD patients. Amongst anti-tTG Ab positive FDRs, high titre (>5 times and 3–4 times above the cut-off value for the positive test) of anti-tTG Ab was more common in females than males (Table 2). 3.3. Association between symptoms and anti-tTG Ab status FDRs with positive anti-tTG Ab test were more frequently symptomatic (p = 0.044), and extra-intestinal symptoms were significantly more common in anti-tTG Ab-positive than negative anti-tTG Ab FDRs (p = 0.004). Amongst gastro-intestinal symptoms, vomiting was more common in anti-tTG Ab positive FDRs than those with a negative anti-tTG Ab test (p = 0.044). Amongst nongastrointestinal symptoms, failure to gain weight was significantly
Please cite this article in press as: Mishra A, et al. Prevalence of celiac disease among first-degree relatives of Indian celiac disease patients. Dig Liver Dis (2015), http://dx.doi.org/10.1016/j.dld.2015.11.007
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Table 1 Symptoms of first-degree relatives of celiac disease patients based on anti-tissue transglutaminase antibody status. Symptoms
Total(n = 434)
Gastrointestinal Diarrhoea Constipation Abdominal pain Vomiting Extraintestinal Arthralgia Anaemia Generalized weakness Oral ulcer Failure to gain weight Failure to gain height
140 (32.2%) 27 (6.2%) 58 (13.3%) 67 (15.4%) 32 (7.4%) 253 (58.3%) 116 (26.7%) 146 (33.6%) 151 (34.8%) 57 (13.1%) 61 (14.0%) 19 (4.3%)
Anti-tTG Ab
p value
Negative (n = 376)
Positive (n = 58)
113 (30%) 22 (5.8%) 51 (13.5%) 54 (14.3%) 24 (6.3%) 209 (55.3%) 101 (26.8%) 122 (32.4%) 126 (33.5%) 49 (13.0%) 40 (10.6%) 15 (3.9%)
27 (46.5%) 5 (8.6%) 7 (12.0%) 13 (22.4%) 8 (13.7%) 44 (75.8%) 15 (25.8%) 24 (41.3%) 25 (43.1%) 8 (13.7%) 21 (36.2%) 4 (6.8%)
0.01 0.41 0.75 0.11 0.04 0.004 0.87 0.18 0.15 0.78 <0.001 0.31
Anti-tTG Ab, anti-tissue transglutaminase antibody.
more common in anti-tTG Ab positive FDRs than those with a negative anti-tTG Ab (p < 0.001; Table 1). 3.4. Grades of villous abnormalities Overall only 28 seropositive FDRs (48.3%) agreed to undergo upper endoscopy and further tests including duodenal biopsy; 21 had villous abnormalities of modified Marsh grade 2 or more (75%: grade 2, n = 2; grade 3a, n = 6; grade 3b, n = 3; grade 3c, n = 10) on histological analysis of duodenal mucosal biopsies. The remaining 25% had villous abnormalities of modified Marsh grade 0 (n = 5) and grade 1 (n = 2; Fig. 1) and were labelled as having potential CD.
We presumed that also 75% of FDRs who did not undergo endoscopic examination might have some form of villous abnormalities of modified Marsh grade 2 or more; estimating 23 cases of CD among FDRs who did not undergo endoscopy, plus 21 cases of histologically confirmed CD and 4 previous diagnoses of CD, the overall prevalence of CD in our cohort of FDRs would be 10.9%. All FDRs who were diagnosed with CD were counselled for gluten-free diet by a nutritionist. All were prescribed calcium and vitamin D3 supplements.
234 families were contacted from the a cohort of 540 celiac disease patients
3.5. Symptoms and classification of CD in FDRs Overall 21 FDRs were diagnosed as CD based on a positive antitTG Ab and villous abnormalities of modified Marsh grade 2 or more. Classical symptoms of CD were present in 12 FDRs (diarrhoea and anaemia, n = 1; anaemia, n = 11; failure to gain in weight, n = 10). Atypical symptoms of CD were present in 7 FDRs (arthralgia, n = 3; constipation, n = 5; vomiting, n = 3; recurrent oral ulcer, n = 3; failure to gain height, n = 1). Two FDRs with CD were completely asymptomatic.
Families agreed to participate (n=176)
Total family members screened (n=434)
3.6. Overall prevalence of CD in FDRs Of 58 anti-tTG Ab positive FDRs, 28 underwent endoscopic examination while 30 declined (Fig. 1). Of these, 21 FDRs (75%) showed villous abnormalities of modified Marsh grade 2 or more suggesting a diagnosis of CD. We compared the characteristics of those who underwent evaluation of villous changes and those who did not (Table 3). While there were no differences in the demographic characteristics and the titre of anti-tTG Ab between these two groups, constipation (p = 0.026) and anaemia (p = 0.02) were more frequent in those who underwent endoscopic examination.
Anti-tissue transglutaminase antibody negative (n=376) 86.7%
Male Female Total
Anti-tTG Ab negative
214 (91.4%) 162 (81.0%) 376 (86.7%)
Anti-tTG Ab positive (titre) (N = 58) 1–2x
3–4x
≥5x
5 (2.1%) 16 (8%) 21 (4.8%)
2 (0.9%) 5 (2.5%) 7 (1.6%)
13 (5.5%) 17 (8.5%) 30 (6.9%)
Anti-tTG Ab, anti-tissue transglutaminase antibody.
Total
234 200 434
Anti-tissue transglutaminase antibody positive (n=58) 13.3%
Endoscopy and biopsy done (n=28)
Table 2 Pattern of anti-tissue transglutaminase antibody titre in 434 first-degree relatives of celiac disease patients. Gender
Asymptomatic (n=152) 35.0% Symptomatic (n=282) 64.9%
An-ssue transglutaminase Ab (IgA)
Endoscopy and biopsy Could not be done ((n=30)
Villous abnormality using Modified Marsh grading
p value
0.007
Potential Celiac disease (n=7) Marsh grade 0 (n=5) Marsh grade 1 (n=2)
Celiac disease (n=21) Marsh grade 2 (n=2) Marsh grade 3 (n=19)
Fig. 1. Flow chart showing recruitment, seroprevalence and prevalence of celiac disease in first-degree relatives of patients with celiac disease. Ab, antibody.
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Table 3 Comparison of symptoms between first-degree relatives with confirmed celiac disease and anti-tissue transglutaminase antibody-positive cases who did not undergo histological examination (refused upper endoscopy).
Age Anti-tTG Ab titre Diarrhoea Constipation Abdominal pain Anaemia Vomiting Failure to gain height Failure to gain weight Arthralgia Generalized weakness Oral ulcer
Confirmed CD (n = 21)
Anti-tTG Ab-positive, no endoscopy (n = 30)
P
31.2 ± 15.5 124.43 ± 92.22 1 (4.8%) 5 (23.3%) 0 (0%) 12 (57.1%) 3 (14.3%) 1 (4.8%) 10 (47.6%) 3 (14.3%) 10 (47.6%) 3 (14.3%)
29.63 ± 18.154 129.64 ± 104.07 2 (6.7%) 1 (3.3%) 1 (3.3%) 8 (26.7%) 3 (10%) 3 (10%) 9 (30%) 5 (16.7%) 9 (30%) 5 (16.7%)
0.21 0.24 0.77 0.026 0.46 0.02 0.64 0.49 0.20 0.818 0.20 0.82
Anti-tTG Ab, anti-tissue transglutaminase antibody.
Of 21 FDRs with CD, 18 accepted GFD and at 6-months of follow up, all of them showed improvement. Three of them did not accept GFD. 3.7. Prevalence of HLA-DQ2 and -DQ8 Haplotyping for HLA-DQ2 and -DQ8 was performed in 127 FDRs (29.3%). HLA-DQ2 and -DQ8 were present in 111 (87.4%); 93 had HLA-DQ2 haplotype (73%), 13 had HLA-DQ8 (10.2%), and 5 had both HLA-DQ2 and-DQ8 haplotype (4%). Among FDRs with HLA-DQ2 and -DQ8 haplotypes, 78 were carrying only one allele of HLA-DQ2 (heterozygous HLA-DQ2, 61.4%), 15 were homozygous HLA-DQ2 (11.8%), 12 were heterozygous HLA-DQ8 (9.4%), and 5 were complex heterozygous carrying alleles for both HLA-DQ2 and HLA-DQ8 (3.9%). Only 1 FDR was homozygous HLA-DQ8 (0.08%). Genotyping for HLA-DQ2 and -DQ8 was performed in 39/58 anti-tTG Ab-positive FDRs. Among them, 28 were HLA-DQ2 heterozygous (71.8%), 8 HLA-DQ2 homozygous (20.5%), 1 HLA-DQ8 heterozygous (2.5%), and 1 had complex hetero DQ2 and DQ8 haplotype (2.5%). One anti-tTG-positive FDR had neither of HLA-DQ2 nor DQ8. 4. Discussion Our study found a seroprevalence of anti-tTG Ab of 13.3% and a prevalence of CD of 10.9% among FDRs of index patients with CD; this is almost 11-fold higher than in the general population. CD in FDRs was more common in females than in males and highest in siblings. Furthermore, 87.4% of FDRs and all but one anti-tTG Abpositive FDRs had haplotype HLA-DQ2 or -DQ8 or both. All FDRs, except two, diagnosed with CD were symptomatic. Because of genetic susceptibility, both first- and second-degree relatives are at a higher risk of developing CD than the general population. The risk of having CD in FDRs varies with the relationship with the index CD patient. Several studies have suggested that siblings are at a higher risk of developing CD than parents and children [19,20,13]. Furthermore, prevalence of CD among FDRs also varies with gender. We also observed that siblings had higher prevalence of CD (sixteen fold higher than the general population) compared to children (5-fold higher than the general population) as also observed by Rubio et al. and Gautam et al. [13,16]. The high prevalence of CD in FDRs (22%) reported by Gautam, et al. is because of inclusion of only siblings in the study [17]. It is therefore important to establish the exact prevalence of CD among FDRs, which is of importance in risk-stratification and screening strategies. The spectrum of phenotypic expression of CD is diverse and extends from completely asymptomatic to severely symptomatic disease [1]. In some, the phenotype of the disease is fully expressed,
in others the disease is expressed only in the milder form [22–24]. In some, the disease manifests clinically in early childhood, while in others the disease becomes apparent at a later age [25]. The spectrum of the symptoms of CD varies from typical symptoms such as diarrhoea, anaemia, abdominal pain and failure to thrive to atypical symptoms such as infertility, osteopenia/osteoporosis [1,10]. Furthermore, many patients with CD may remain completely asymptomatic or have mild symptoms not compelling them to seek medical attention. In the present study, only 57% of FDRs with CD had classical symptoms and 2 were totally asymptomatic. Tursi et al. have also reported 45.8% subclinical symptoms in anti-tTG-positive FDRs [15]. Seven FDRs had potential CD; hence screening of FDRs for CD using serological test is beneficial in identifying silent form of the disease. It is now well established that all FDRs either asymptomatic or symptomatic should be screened for CD. Based on the observation that family members of patients with CD are not only at high risk of developing CD but also are at a higher risk to develop other autoimmune diseases, such as type I diabetes and autoimmune thyroiditis [26,27]. In this study, 3 anti-tTG positive FDRs with CD also had hypothyroidism. Nass et al. observed a significant increase in autoantibodies against thyroid and parietal cells in FDRs compared to controls (p = 0.006) [28]. Such an observation raises an argument in favour of screening FDRs for other autoimmune diseases in addition to CD. Why do FDRs have higher prevalence of CD? The higher risk of CD amongst FDRs can be explained by genetic linkages, one of them being specific HLA haplotypes. One third of general population and 90% of CD patients have HLA-DQ2 or -DQ8 haplotype [29]; 87.4% FDRs also had HLA-DQ2 or -DQ8 haplotype in this study. Similar data was reported in a study conducted by CastroAntunes et al. in 2011 [30]. Srivastava et al. from India in a study including 91 FDRs, also reported that 85.7% of FDRs were carrying HLA–DQ2 haplotype [18]. In addition, FDRs possibly share the same environmental triggers as index patients, which in part are modulated by genetic factors. For example, recent evidence shows that HLA-DQ2 genotype influences the gut microbiota composition of healthy infants at familial risk of CD [31]. Furthermore, while CD occurs due to gluten peptide induced both acquired and innate immune response in a genetically susceptible individual [32], what decides the clinical phenotypic expression is not well known. While presence of HLA haplotype explains approximately 40% of conferred risk, there is a strong likelihood of other genetic factors playing a role in the expression of the disease [33]. One of the well-known factors which affect the clinical expression of the disease is HLA-DQ2 or -DQ8 homozygosity [34–36]. In our cohort all but one anti-tTG Ab-positive FDRs were carrying either HLADQ2 or HLA-DQ8 haplotype. Similar findings were observed by
Please cite this article in press as: Mishra A, et al. Prevalence of celiac disease among first-degree relatives of Indian celiac disease patients. Dig Liver Dis (2015), http://dx.doi.org/10.1016/j.dld.2015.11.007
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Karinen and Martins et al. in a study conducted on HLA genotyping of FDRs [37,38]. Since many FDRs already had suffered from failure to thrive and anaemia, early detection of CD might have prevented growth failure. It is therefore advisable to screen FDRs as early as possible. However, a single serological test may not rule out future development of CD, and the appropriate interval for repeating serological screening is not well established. The strength of the present study is the inclusion of a large number of FDRs, biopsy-based diagnosis of CD and HLA = typing at least in a subset of participants; however there are some weaknesses: only 54% of all FDRs agreed to be screened for CD, hence selection bias cannot be excluded; 65% of FDRs who agreed to be screened were symptomatic, also suggesting a bias. We could not collect the data on the symptoms of FDRs who did not agree to participate in the study. For anti-tTG Ab-positive FDRs who did not undergo histological evaluation the prevalence of CD was derived on assumptions. Furthermore, this study was performed at a tertiary care centre, which might also add to referral bias. Since this study was based on a single test for specific celiac serology and not followed longitudinally, some FDRs may seroconvert in the future, since 87.4% were genetically susceptible. The lower prevalence reported among children of CD patients could be due to small sample size: only 121 children were eligible for screening, of which 84 participated in the study. Another possible bias was significantly younger age of FDRs who participated vs those who did not. Even with the above limitations, our findings confirm that all FDRs of patients with CD should be screened for CD. Conflict of interest None declared. Funding This study is supported by Department of Biotechnology, Ministry of Science and Technology, Government of India. Acknowledgments We are thankful to research staff of Gastroenterology and Human Nutrition for blood collection of family members of celiac disease patients. We are especially thankful to Mr. Satvir, Ms. Chanchal, Mr. Manohar and Mr. Sanjit for their support and help. This study would have been not completed without support of celiac disease patients and their family members. References [1] Ludvigsson JF, Leffler DA, Bai JC, et al. The Oslo definitions for coeliac disease and related terms. Gut 2013;62:43–52. [2] Catassi C, Rätsch IM, Fabiani E, et al. Coeliac disease in the year 2000: exploring the iceberg. Lancet 1994;343:200–3. [3] Barada K, Bitar A, Mokadem MA-R, et al. Celiac disease in Middle Eastern and North African countries: a new burden? World Journal of Gastroenterology 2010;16:1449–57. [4] Fasano A, Berti I, Gerarduzzi T, et al. Prevalence of celiac disease in at-risk and not-at-risk groups in the United States: a large multicenter study. Archives of Internal Medicine 2003;163:286–92. [5] Sood A, Midha V, Sood N, et al. Prevalence of celiac disease among school children in Punjab, North India. Journal of Gastroenterology and Hepatology 2006;21:1622–5. [6] Makharia GK, Verma AK, Amarchand R, et al. Prevalence of celiac disease in the northern part of India: a community based study. Journal of Gastroenterology and Hepatology 2011;26:894–900. [7] Yuan J, Gao J, Li X, et al. The tip of the celiac iceberg in China: a systematic review and meta-analysis. PLoS ONE 2013;8:e81151. [8] Watanabe C, Komoto S, Hokari R, et al. Prevalence of serum celiac antibody in patients with IBD in Japan. Journal of Gastroenterology 2014;49:825–34. [9] Ikram MA, Sajid A, Hameed S, et al. Coeliac disease in children presenting with failure to thrive. Journal of Ayub Medical College Abbottabad 2011;23:6–9.
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Alimentary Tract
Prevalence of celiac disease among first-degree relatives of Indian celiac disease patients Asha Mishra a , Shyam Prakash a , Gurvinder Kaur b , Vishnubhatla Sreenivas c , Vineet Ahuja a , Siddhartha Datta Gupta d , Govind K. Makharia a,∗ a
Department of Gastroenterology and Human Nutrition, All India Institute of Medical Sciences, New Delhi, India Department of Transplant Immunology, All India Institute of Medical Sciences, New Delhi, India c Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India d Department of Pathology, All India Institute of Medical Sciences, New Delhi, India b
a r t i c l e
i n f o
Article history: Received 1 July 2015 Accepted 11 November 2015 Available online xxx Keywords: Family Haplotype HLA Seroprevalence
a b s t r a c t Background: Celiac disease, once thought to be uncommon in Asia, is now recognized in Asian nations as well. We investigated the prevalence of celiac disease in first-degree relatives of celiac disease patients followed in our centre. Methods: First-degree relatives were screened prospectively for celiac disease using questionnaire-based interview and anti-tissue transglutaminase antibody. Serology positive first-degree relatives underwent duodenal biopsies. Diagnosis of celiac disease was made based on positive serology and villous abnormality Marsh grade 2 or higher. Human leucocyte antigen DQ2/-DQ8 was also assessed in 127 first-degree relatives. Results: 434 first-degree relatives of 176 celiac disease patients were prospectively recruited; 282 were symptomatic (64.9%), 58 were positive for serology (13.3%). Seroprevalence was higher in female than in males (19% vs 8.5%; p = 0.001) and highest in siblings (16.9%) than parents (13.6%) and children (5.9%) of celiac patients (p = 0.055); 87.4% first-degree relatives were human leucocyte antigen-DQ2/-DQ8 positive. Overall prevalence of celiac disease was 10.9% amongst first-degree relatives. Conclusions: The prevalence of celiac disease in first-degree relatives of celiac disease patients was 10.9% in our cohort, and 87% had human leucocyte antigen-DQ2 or -DQ8 haplotype. All first-degree relatives of celiac disease patients should be screen for celiac disease even if asymptomatic or with atypical manifestations. © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Celiac disease (CD) is an immune-mediated enteropathy triggered by ingestion of gluten present in wheat and related prolamines present in rye and barley in genetically susceptible individuals carrying HLA-DQ2 and/or HLA-DQ8 haplotype [1]. Once thought to be uncommon, CD affects approximately 1% of the world’s population [2–4]. Even in Asian countries, where CD was thought to be a rare disease, recent studies – including one from our centre – suggest a prevalence of 1 in 330 to 1 in 96 in the general population in Northern India [5,6]. CD is now being observed
∗ Corresponding author at: Department of Gastroenterology and Human Nutrition, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. Tel.: +91 11 26588091/26546546; fax: +91 11 26588091/26588663. E-mail addresses: [email protected], [email protected] (G.K. Makharia).
in other Asian countries including China, Malaysia and Pakistan [7–10]. Since CD is a genetic disease, first-degree relatives (FDRs) of patients with CD are at higher risk of developing CD due to close genetic repertoire, which leads to higher genetic susceptibility [4]. Advent of celiac-specific antibodies (a reflection of adaptive immune response to gluten peptide) has revolutionized the case detection rate and has led to recognition of CD as a public health problem world over [11]. With the help of celiac-specific serologic tests, it is now possible to screen and detect CD not only the clinically apparent patients but also those who still have not developed any symptoms. With increasing use of screening and diagnostic tests along with increase in the awareness, CD has become one of the most common genetic disease. Because of the genetic susceptibility, both FDRs and seconddegree relatives are at a higher risk of developing CD than the general population. The prevalence of CD in FDRs vary widely from 5% to 38% [12–15]. The three studies from Asia, all the them from
http://dx.doi.org/10.1016/j.dld.2015.11.007 1590-8658/© 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
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the Northern part of India have also reported a prevalence of CD in FDRs to vary from 8.2% to 22% [16–18]. The risk of having CD in FDRs varies with the individual relationship with the index patient with CD. Several studies have suggested that siblings are at a higher risk of developing CD in comparison to parents and children [19,20,13]. The spectrum of symptoms in FDRs varies and a large number of them remain either asymptomatic and or mildly symptomatic. Only a proportion of them have full-blown symptoms. Since CD is now emerging in Asian countries, and there are two other studies from India with much smaller sample size in both the studies and inclusion of only siblings in one of the study [16,17]. Therefore, we conducted the present study to find out the prevalence of CD in the FDRs of patients with CD. 2. Methods 2.1. Subjects This prospective study was conducted at All India Institute of Medical Sciences, New Delhi, between May 2009 and September 2014. From the initial cohort of 540 patients with CD followed at our Celiac Disease Clinic, all FDRs were invited to be screened for CD. Four hundred and thirty four FDRs (54% of total live FDRs) of 176 index patients agreed to participate and were enrolled. This study was approved by the Ethics Committee of our Institution and written informed consent was obtained from each participant and parents or guardians for subjects aged less than 18 years. All FDRs were administered a questionnaire and they underwent a complete history and clinical evaluation. Five-millilitres of blood was collected and separated in a plain tube (2 ml) and in EDTA (3 ml). Plain tube sample was kept at room temperature for approximately an hour and then centrifuged at 2500 × g for 10 min. Supernatant was separated and was kept in a microcentrifuge tube at 20 ◦ C until analysis. DNA was isolated from the blood collected in the EDTA tubes. 2.2. IgA anti-tissue transglutaminase antibody (IgA anti-tTG Ab) FDRs were screened for CD using commercially available IgA anti-tTG Ab ELISA kits procured from AESKU Diagnostik, Wendelsheim, Germany (cut-off: 18 IU/ml). FDRs with a positive ant-tTG Ab were invited to undergo upper gastrointestinal endoscopy and duodenal biopsies.
The diagnosis of CD was made on the basis of the modified European Society of Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) criteria i.e., clinical features, presence of villous atrophy (villous abnormalities of modified Marsh grade ≥2) and unequivocal response to gluten-free diet [21]. 2.4. HLA genotyping The genotyping for HLA-DQ2 and -DQ8 was performed using reverse sequence specific oligonucleotides method (One Lambda, Inc., Thermo Fisher Scientific, USA). Readings were taken through Luminex X-Map technology (Luminex, Life Technologies, Inc). 2.5. Statistical analysis Quantitative variables were summarized as mean ± standard deviation. Qualitative were summarized as frequency (%). Qualitative variables were compared between those with a positive and negative anti-tTG Ab using Chi-square test. Quantitative variables were compared using student’s t test or Wilcoxon’s Ranksum test as appropriate. 3. Results Overall, 434 FDRs of 176 CD patients (54% of all living FDRs) were included in this study; 234 (53.9%) were males, mean age was 29.8 ± 15.9 years; 326 were over 18 years of age (75.1%). Relationships with patients were 159 siblings (36.6%), 191 parents (44%), and 84 children (19.3%). Four FDRs had previously been diagnosed with CD and were already following gluten-free diet. FDRs who agreed to participate were younger than those who did not consent (29.71 ± 15.95 vs 37.38 ± 16.7 years). However, there was no difference in gender distribution. 3.1. Symptoms Two hundred and eighty two FDRs (64.9%) had one or more symptoms; 140 had predominantly gastrointestinal symptoms (32.2%) and 253 had a combination of both gastrointestinal and extra intestinal symptoms (58.2%; Table 1); 152 were completely asymptomatic (35%). Hypothyroidism was present in 4. 3.2. Seroprevalence of CD
2.3. Endoscopy and mucosal biopsy examination Upper gastrointestinal endoscopy was performed and at least four to six biopsies were taken from the second or third part of the duodenum. A biopsy fragment was considered oriented when at least 3 crypts were oriented perpendicularly on the underlying muscularis mucosae. Biopsies were analyzed for mucosal changes by a histo-pathologist with special expertise in gastrointestinal pathology blinded to the clinical or serological results. The Modified Marsh grading system was used for grading mucosal changes: Grade 0, normal histology [a crypt to villous ratio of 1:3 was taken as normal]; Grade 1:increase of intraepithelial lymphocytes (IEL) >40/100 enterocytes (IELs were identified as dark round cells with high nucleus to cytoplasmic ratio, in comparison to the perpendicularly oriented cigar shaped vesicular nuclei of the mucosal epithelial cells); Grade 2: increased IELs along with crypt hyperplasia; Grade 3: increased IELs along with crypt hyperplasia and variable degrees of villous atrophy. A further semi-quantitative sub typing of the villous atrophy was performed as follows: grade 3a/mild villous atrophy [duodenal biopsies with a crypt to villous ration of >1:3 but <1]; grade 3b/moderate villous atrophy, C:V ratio of 1:1; grade 3c/severe villous atrophy, C:V ratio of >1.
Overall, 58 FDRs belonging to 44 index patient families tested positive for anti-tTG Ab (13.3%). This meant approximately 25% (one in 4) of families had at least one anti-tTG Ab positive FDR. Five families of index CD patients had 2 or more anti-tTG Ab positive FDRs. CD seroprevalence was higher in female than in male FDRs (19% vs 8.5%; p = 0.001). The seroprevalence was 16.9% in siblings vs 13.6% in parents and 5.9% in children (p = 0.055) of CD patients. Amongst anti-tTG Ab positive FDRs, high titre (>5 times and 3–4 times above the cut-off value for the positive test) of anti-tTG Ab was more common in females than males (Table 2). 3.3. Association between symptoms and anti-tTG Ab status FDRs with positive anti-tTG Ab test were more frequently symptomatic (p = 0.044), and extra-intestinal symptoms were significantly more common in anti-tTG Ab-positive than negative anti-tTG Ab FDRs (p = 0.004). Amongst gastro-intestinal symptoms, vomiting was more common in anti-tTG Ab positive FDRs than those with a negative anti-tTG Ab test (p = 0.044). Amongst nongastrointestinal symptoms, failure to gain weight was significantly
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Table 1 Symptoms of first-degree relatives of celiac disease patients based on anti-tissue transglutaminase antibody status. Symptoms
Total(n = 434)
Gastrointestinal Diarrhoea Constipation Abdominal pain Vomiting Extraintestinal Arthralgia Anaemia Generalized weakness Oral ulcer Failure to gain weight Failure to gain height
140 (32.2%) 27 (6.2%) 58 (13.3%) 67 (15.4%) 32 (7.4%) 253 (58.3%) 116 (26.7%) 146 (33.6%) 151 (34.8%) 57 (13.1%) 61 (14.0%) 19 (4.3%)
Anti-tTG Ab
p value
Negative (n = 376)
Positive (n = 58)
113 (30%) 22 (5.8%) 51 (13.5%) 54 (14.3%) 24 (6.3%) 209 (55.3%) 101 (26.8%) 122 (32.4%) 126 (33.5%) 49 (13.0%) 40 (10.6%) 15 (3.9%)
27 (46.5%) 5 (8.6%) 7 (12.0%) 13 (22.4%) 8 (13.7%) 44 (75.8%) 15 (25.8%) 24 (41.3%) 25 (43.1%) 8 (13.7%) 21 (36.2%) 4 (6.8%)
0.01 0.41 0.75 0.11 0.04 0.004 0.87 0.18 0.15 0.78 <0.001 0.31
Anti-tTG Ab, anti-tissue transglutaminase antibody.
more common in anti-tTG Ab positive FDRs than those with a negative anti-tTG Ab (p < 0.001; Table 1). 3.4. Grades of villous abnormalities Overall only 28 seropositive FDRs (48.3%) agreed to undergo upper endoscopy and further tests including duodenal biopsy; 21 had villous abnormalities of modified Marsh grade 2 or more (75%: grade 2, n = 2; grade 3a, n = 6; grade 3b, n = 3; grade 3c, n = 10) on histological analysis of duodenal mucosal biopsies. The remaining 25% had villous abnormalities of modified Marsh grade 0 (n = 5) and grade 1 (n = 2; Fig. 1) and were labelled as having potential CD.
We presumed that also 75% of FDRs who did not undergo endoscopic examination might have some form of villous abnormalities of modified Marsh grade 2 or more; estimating 23 cases of CD among FDRs who did not undergo endoscopy, plus 21 cases of histologically confirmed CD and 4 previous diagnoses of CD, the overall prevalence of CD in our cohort of FDRs would be 10.9%. All FDRs who were diagnosed with CD were counselled for gluten-free diet by a nutritionist. All were prescribed calcium and vitamin D3 supplements.
234 families were contacted from the a cohort of 540 celiac disease patients
3.5. Symptoms and classification of CD in FDRs Overall 21 FDRs were diagnosed as CD based on a positive antitTG Ab and villous abnormalities of modified Marsh grade 2 or more. Classical symptoms of CD were present in 12 FDRs (diarrhoea and anaemia, n = 1; anaemia, n = 11; failure to gain in weight, n = 10). Atypical symptoms of CD were present in 7 FDRs (arthralgia, n = 3; constipation, n = 5; vomiting, n = 3; recurrent oral ulcer, n = 3; failure to gain height, n = 1). Two FDRs with CD were completely asymptomatic.
Families agreed to participate (n=176)
Total family members screened (n=434)
3.6. Overall prevalence of CD in FDRs Of 58 anti-tTG Ab positive FDRs, 28 underwent endoscopic examination while 30 declined (Fig. 1). Of these, 21 FDRs (75%) showed villous abnormalities of modified Marsh grade 2 or more suggesting a diagnosis of CD. We compared the characteristics of those who underwent evaluation of villous changes and those who did not (Table 3). While there were no differences in the demographic characteristics and the titre of anti-tTG Ab between these two groups, constipation (p = 0.026) and anaemia (p = 0.02) were more frequent in those who underwent endoscopic examination.
Anti-tissue transglutaminase antibody negative (n=376) 86.7%
Male Female Total
Anti-tTG Ab negative
214 (91.4%) 162 (81.0%) 376 (86.7%)
Anti-tTG Ab positive (titre) (N = 58) 1–2x
3–4x
≥5x
5 (2.1%) 16 (8%) 21 (4.8%)
2 (0.9%) 5 (2.5%) 7 (1.6%)
13 (5.5%) 17 (8.5%) 30 (6.9%)
Anti-tTG Ab, anti-tissue transglutaminase antibody.
Total
234 200 434
Anti-tissue transglutaminase antibody positive (n=58) 13.3%
Endoscopy and biopsy done (n=28)
Table 2 Pattern of anti-tissue transglutaminase antibody titre in 434 first-degree relatives of celiac disease patients. Gender
Asymptomatic (n=152) 35.0% Symptomatic (n=282) 64.9%
An-ssue transglutaminase Ab (IgA)
Endoscopy and biopsy Could not be done ((n=30)
Villous abnormality using Modified Marsh grading
p value
0.007
Potential Celiac disease (n=7) Marsh grade 0 (n=5) Marsh grade 1 (n=2)
Celiac disease (n=21) Marsh grade 2 (n=2) Marsh grade 3 (n=19)
Fig. 1. Flow chart showing recruitment, seroprevalence and prevalence of celiac disease in first-degree relatives of patients with celiac disease. Ab, antibody.
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Table 3 Comparison of symptoms between first-degree relatives with confirmed celiac disease and anti-tissue transglutaminase antibody-positive cases who did not undergo histological examination (refused upper endoscopy).
Age Anti-tTG Ab titre Diarrhoea Constipation Abdominal pain Anaemia Vomiting Failure to gain height Failure to gain weight Arthralgia Generalized weakness Oral ulcer
Confirmed CD (n = 21)
Anti-tTG Ab-positive, no endoscopy (n = 30)
P
31.2 ± 15.5 124.43 ± 92.22 1 (4.8%) 5 (23.3%) 0 (0%) 12 (57.1%) 3 (14.3%) 1 (4.8%) 10 (47.6%) 3 (14.3%) 10 (47.6%) 3 (14.3%)
29.63 ± 18.154 129.64 ± 104.07 2 (6.7%) 1 (3.3%) 1 (3.3%) 8 (26.7%) 3 (10%) 3 (10%) 9 (30%) 5 (16.7%) 9 (30%) 5 (16.7%)
0.21 0.24 0.77 0.026 0.46 0.02 0.64 0.49 0.20 0.818 0.20 0.82
Anti-tTG Ab, anti-tissue transglutaminase antibody.
Of 21 FDRs with CD, 18 accepted GFD and at 6-months of follow up, all of them showed improvement. Three of them did not accept GFD. 3.7. Prevalence of HLA-DQ2 and -DQ8 Haplotyping for HLA-DQ2 and -DQ8 was performed in 127 FDRs (29.3%). HLA-DQ2 and -DQ8 were present in 111 (87.4%); 93 had HLA-DQ2 haplotype (73%), 13 had HLA-DQ8 (10.2%), and 5 had both HLA-DQ2 and-DQ8 haplotype (4%). Among FDRs with HLA-DQ2 and -DQ8 haplotypes, 78 were carrying only one allele of HLA-DQ2 (heterozygous HLA-DQ2, 61.4%), 15 were homozygous HLA-DQ2 (11.8%), 12 were heterozygous HLA-DQ8 (9.4%), and 5 were complex heterozygous carrying alleles for both HLA-DQ2 and HLA-DQ8 (3.9%). Only 1 FDR was homozygous HLA-DQ8 (0.08%). Genotyping for HLA-DQ2 and -DQ8 was performed in 39/58 anti-tTG Ab-positive FDRs. Among them, 28 were HLA-DQ2 heterozygous (71.8%), 8 HLA-DQ2 homozygous (20.5%), 1 HLA-DQ8 heterozygous (2.5%), and 1 had complex hetero DQ2 and DQ8 haplotype (2.5%). One anti-tTG-positive FDR had neither of HLA-DQ2 nor DQ8. 4. Discussion Our study found a seroprevalence of anti-tTG Ab of 13.3% and a prevalence of CD of 10.9% among FDRs of index patients with CD; this is almost 11-fold higher than in the general population. CD in FDRs was more common in females than in males and highest in siblings. Furthermore, 87.4% of FDRs and all but one anti-tTG Abpositive FDRs had haplotype HLA-DQ2 or -DQ8 or both. All FDRs, except two, diagnosed with CD were symptomatic. Because of genetic susceptibility, both first- and second-degree relatives are at a higher risk of developing CD than the general population. The risk of having CD in FDRs varies with the relationship with the index CD patient. Several studies have suggested that siblings are at a higher risk of developing CD than parents and children [19,20,13]. Furthermore, prevalence of CD among FDRs also varies with gender. We also observed that siblings had higher prevalence of CD (sixteen fold higher than the general population) compared to children (5-fold higher than the general population) as also observed by Rubio et al. and Gautam et al. [13,16]. The high prevalence of CD in FDRs (22%) reported by Gautam, et al. is because of inclusion of only siblings in the study [17]. It is therefore important to establish the exact prevalence of CD among FDRs, which is of importance in risk-stratification and screening strategies. The spectrum of phenotypic expression of CD is diverse and extends from completely asymptomatic to severely symptomatic disease [1]. In some, the phenotype of the disease is fully expressed,
in others the disease is expressed only in the milder form [22–24]. In some, the disease manifests clinically in early childhood, while in others the disease becomes apparent at a later age [25]. The spectrum of the symptoms of CD varies from typical symptoms such as diarrhoea, anaemia, abdominal pain and failure to thrive to atypical symptoms such as infertility, osteopenia/osteoporosis [1,10]. Furthermore, many patients with CD may remain completely asymptomatic or have mild symptoms not compelling them to seek medical attention. In the present study, only 57% of FDRs with CD had classical symptoms and 2 were totally asymptomatic. Tursi et al. have also reported 45.8% subclinical symptoms in anti-tTG-positive FDRs [15]. Seven FDRs had potential CD; hence screening of FDRs for CD using serological test is beneficial in identifying silent form of the disease. It is now well established that all FDRs either asymptomatic or symptomatic should be screened for CD. Based on the observation that family members of patients with CD are not only at high risk of developing CD but also are at a higher risk to develop other autoimmune diseases, such as type I diabetes and autoimmune thyroiditis [26,27]. In this study, 3 anti-tTG positive FDRs with CD also had hypothyroidism. Nass et al. observed a significant increase in autoantibodies against thyroid and parietal cells in FDRs compared to controls (p = 0.006) [28]. Such an observation raises an argument in favour of screening FDRs for other autoimmune diseases in addition to CD. Why do FDRs have higher prevalence of CD? The higher risk of CD amongst FDRs can be explained by genetic linkages, one of them being specific HLA haplotypes. One third of general population and 90% of CD patients have HLA-DQ2 or -DQ8 haplotype [29]; 87.4% FDRs also had HLA-DQ2 or -DQ8 haplotype in this study. Similar data was reported in a study conducted by CastroAntunes et al. in 2011 [30]. Srivastava et al. from India in a study including 91 FDRs, also reported that 85.7% of FDRs were carrying HLA–DQ2 haplotype [18]. In addition, FDRs possibly share the same environmental triggers as index patients, which in part are modulated by genetic factors. For example, recent evidence shows that HLA-DQ2 genotype influences the gut microbiota composition of healthy infants at familial risk of CD [31]. Furthermore, while CD occurs due to gluten peptide induced both acquired and innate immune response in a genetically susceptible individual [32], what decides the clinical phenotypic expression is not well known. While presence of HLA haplotype explains approximately 40% of conferred risk, there is a strong likelihood of other genetic factors playing a role in the expression of the disease [33]. One of the well-known factors which affect the clinical expression of the disease is HLA-DQ2 or -DQ8 homozygosity [34–36]. In our cohort all but one anti-tTG Ab-positive FDRs were carrying either HLADQ2 or HLA-DQ8 haplotype. Similar findings were observed by
Please cite this article in press as: Mishra A, et al. Prevalence of celiac disease among first-degree relatives of Indian celiac disease patients. Dig Liver Dis (2015), http://dx.doi.org/10.1016/j.dld.2015.11.007
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Karinen and Martins et al. in a study conducted on HLA genotyping of FDRs [37,38]. Since many FDRs already had suffered from failure to thrive and anaemia, early detection of CD might have prevented growth failure. It is therefore advisable to screen FDRs as early as possible. However, a single serological test may not rule out future development of CD, and the appropriate interval for repeating serological screening is not well established. The strength of the present study is the inclusion of a large number of FDRs, biopsy-based diagnosis of CD and HLA = typing at least in a subset of participants; however there are some weaknesses: only 54% of all FDRs agreed to be screened for CD, hence selection bias cannot be excluded; 65% of FDRs who agreed to be screened were symptomatic, also suggesting a bias. We could not collect the data on the symptoms of FDRs who did not agree to participate in the study. For anti-tTG Ab-positive FDRs who did not undergo histological evaluation the prevalence of CD was derived on assumptions. Furthermore, this study was performed at a tertiary care centre, which might also add to referral bias. Since this study was based on a single test for specific celiac serology and not followed longitudinally, some FDRs may seroconvert in the future, since 87.4% were genetically susceptible. The lower prevalence reported among children of CD patients could be due to small sample size: only 121 children were eligible for screening, of which 84 participated in the study. Another possible bias was significantly younger age of FDRs who participated vs those who did not. Even with the above limitations, our findings confirm that all FDRs of patients with CD should be screened for CD. Conflict of interest None declared. Funding This study is supported by Department of Biotechnology, Ministry of Science and Technology, Government of India. Acknowledgments We are thankful to research staff of Gastroenterology and Human Nutrition for blood collection of family members of celiac disease patients. We are especially thankful to Mr. Satvir, Ms. Chanchal, Mr. Manohar and Mr. Sanjit for their support and help. This study would have been not completed without support of celiac disease patients and their family members. References [1] Ludvigsson JF, Leffler DA, Bai JC, et al. The Oslo definitions for coeliac disease and related terms. Gut 2013;62:43–52. [2] Catassi C, Rätsch IM, Fabiani E, et al. Coeliac disease in the year 2000: exploring the iceberg. Lancet 1994;343:200–3. [3] Barada K, Bitar A, Mokadem MA-R, et al. Celiac disease in Middle Eastern and North African countries: a new burden? World Journal of Gastroenterology 2010;16:1449–57. [4] Fasano A, Berti I, Gerarduzzi T, et al. Prevalence of celiac disease in at-risk and not-at-risk groups in the United States: a large multicenter study. Archives of Internal Medicine 2003;163:286–92. [5] Sood A, Midha V, Sood N, et al. Prevalence of celiac disease among school children in Punjab, North India. Journal of Gastroenterology and Hepatology 2006;21:1622–5. [6] Makharia GK, Verma AK, Amarchand R, et al. Prevalence of celiac disease in the northern part of India: a community based study. Journal of Gastroenterology and Hepatology 2011;26:894–900. [7] Yuan J, Gao J, Li X, et al. The tip of the celiac iceberg in China: a systematic review and meta-analysis. PLoS ONE 2013;8:e81151. [8] Watanabe C, Komoto S, Hokari R, et al. Prevalence of serum celiac antibody in patients with IBD in Japan. Journal of Gastroenterology 2014;49:825–34. [9] Ikram MA, Sajid A, Hameed S, et al. Coeliac disease in children presenting with failure to thrive. Journal of Ayub Medical College Abbottabad 2011;23:6–9.
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Please cite this article in press as: Mishra A, et al. Prevalence of celiac disease among first-degree relatives of Indian celiac disease patients. Dig Liver Dis (2015), http://dx.doi.org/10.1016/j.dld.2015.11.007